Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellular lipid kinases that phosphorylate the 3-position hydroxyl group of the inositol ring of phosphoinositide lipids (PIs) generating lipid second messengers. While α and β isoforms of PI3K are ubiquitous in their distribution, expression of δ and γ forms of PI3K is restricted to circulating haematogenous cells and endothelial cells. Unlike PI3Kα or PI3Kβ, mice lacking expression of PI3Kδ or PI3Kγ do not show any adverse phenotype indicating that targeting of these specific isoforms would not result in overt toxicity.
Recently, targeted inhibitors of the PI3K pathway have been suggested as immunomodulatory agents. This interest stems from the fact that the PI3K pathway serves multiple functions in immune cell signaling, primarily through the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a membrane bound second messenger. PIP3 recruits proteins to the cytoplasmic side of the lipid bilayer, including protein kinases and GTPases, initiating a complex network of downstream signaling cascades important in the regulation of immune cell adhesion, migration, and cell-cell communication.
The four class I PI3K isoforms differ significantly in their tissue distribution. PI3Kα and PI3Kβ are ubiquitous and activated downstream of receptor tyrosine kinases (RTK), whereas PI3Kδ and PI3Kγ are primarily limited to hematopoietic and endothelial cells, and are activated downstream of RTKs, and G protein coupled receptors (GPCR), respectively. Mouse genetic studies have revealed that PI3Kα and PI3Kβ are essential for normal development, whereas loss of PI3Kδ and/or PI3Kγ yields viable offspring with selective immune deficits.
The expression pattern and functions of PI3Kδ and PI3Kγ have generated much interest in developing PI3Kδ/γ inhibitors as active agents for the treatment of many diseases, including, for example, rheumatoid arthritis, allergies, asthma, chronic obstructive pulmonary disease and multiple sclerosis (Hirsch et al., Pharmacol. Ther., 118, 192-205, 2008; Marone et al., Biochim. Biophys. Acta., 1784, 159-185, 2008; Rommel et al., Nat. Rev. Immunol., 7, 191-201, 2007; Ruckle et al., Nat. Rev. Drug Discov., 5, 903-918, 2006). Studies using both pharmacologic and genetic methods have shown these two isoforms often demonstrate synergistic interactions with each other (Konrad et al., J. Biol. Chem., 283, 33296-33303, 2008; Laffargue et al., Immunity, 16, 441-451, 2002). In mast cells, for example, PI3Kδ is essential for degranulation in response to IgE cross-linking of Fc-receptors (Ali et al., J. Immunol., 180, 2538-2544, 2008), while PI3Kγ plays an important role in amplifying the response (Laffargue et al., Immunity, 16, 441-451, 2002). Similar effects have been seen in other cellular functions, including lymphocyte homing and the neutrophil respiratory burst where PI3Kγ plays a critical role and PI3Kδ amplifies each process. The nonredundant but related roles of PI3Kδ and PI3Kγ have made it difficult to determine which of the two isoforms (alone or in combination) is best targeted in a particular inflammatory disorder.
Studies using mice that lack PI3Kδ and/or PI3Kγ or express kinase-dead variants of PI3Kδ and PI3Kγ have been valuable tools in understanding their roles. For example, PI3Kδ knockout mice demonstrated diminished neutrophil chemotaxis, diminished antibody production (both T cell dependent and independent) (Jou et al., Mol. Cell. Biol., 22, 8580-8591, 2002), and lower numbers of mature B cells (Clayton et al., J. Exp. Med., 196, 753-763, 2002; Jou et al., Mol. Cell. Biol., 22, 8580-8591, 2002), and a decrease in their proliferation in response to anti-IgM (Jou et al., Mol. Cell. Biol., 22, 8580-8591, 2002). This phenotype was replicated in the PI3Kδ kinase-dead variant and with PI3Kδ selective inhibitors along with a decreased number and proliferation of mast cells, and an attenuated allergic response. The PI3Kγ knockout contained higher numbers of, but less responsive, neutrophils, lower numbers of and less responsive macrophages, and dendritic cells displayed decreased mast cell degranulation (Laffargue et al., Immunity, 16, 441-451, 2002), a higher ratio of CD4+ to CD8+ T cells, increased thymocyte apoptosis, diminished induction of CXCR3 on activated T cells and decreased cardiac contractility. This latter effect on cardiac tissue was a concern for chronic dosing of patients with PI3Kγ inhibitors. However, this concern was largely mitigated when the PI3Kγ kinase-dead variant (which better mimics inhibition of the kinase rather than loss of the protein) showed similar immune cell phenotypes, but importantly had no cardiac defects. The cardiac effect was later shown to be due to scaffolding effects rather than the catalytic activity of PI3Kγ (Olusegon et al., Chemistry & Biology, 1, 123-134, 2010, including the references cited therein). The dual PI3Kδ/PI3Kγ knockout was viable but exhibited serious defects in T cell development and thymocyte survival. The PI3Kγ knockout/PI3Kδ kinase-dead combination produced a similar phenotype suggesting that at least within the immune system, the role of PI3Kδ is likely only a catalytic one. Interpretation of studies using knockout and kinase-dead mice can be challenging because these models provide only a steady-state picture of the immune system, lack temporal and dose control, and do not permit a full understanding of how a dynamic immune response will react to reversible inhibition. Selective inhibitors with varying profiles (PI3Kδ, PI3Kγ, and PI3Kδ/γ) are necessary for studies of leukocyte signaling in order to assess the relative contributions of each PI3K to immune cell activation (Olusegon et al., supra, including the references cited therein).
Dual inhibition of δ/γ is strongly implicated as an intervention strategy in allergic and non-allergic inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3Kδ and PI3K γ involvement in various cellular processes underlying asthma and chronic obstructive pulmonary disease (COPD) stems from inhibitor studies and gene-targeting approaches (William et. al Chemistry & Biology, 17, 123-134, 2010 and Thompson, et al. Chemistry & Biology, 17:101-102, 2010). Also, resistance to conventional therapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K δ/γ pathway. Disruption of PI3Kδ/γ signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotal role played by PI3Kδ and PI3Kγ in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well. Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the PI3Kδ and PI3Kγ isoforms would be expected to attenuate the progression of immune response encountered in airway inflammation and rheumatoid arthritis (William et. al Chemistry & Biology, 17, 123-134, 2010 and Thompson, et al. Chemistry & Biology, 17:101-102, 2010)
Reviews and studies regarding PI3K and related protein kinase pathways have been given by Liu et al., Nature Reviews Drug Discovery, 8, 627-644, 2009); Nathan et. al., Mol Cancer Ther., 8(1), 2009; Marone et al., Biochimica et Biophysica Acta, 1784, 159-185, 2008 and Markman et al., Annals of Oncology Advance Access, published August 2009. Similarly reviews and studies regarding role of PI3Kδ and PI3Kγ have been given by William et al., Chemistry & Biology, 17, 123-134, 2010 and Timothy et al. J. Med. Chem., 55 (20), 8559-8581, 2012. All of these literature disclosures are hereby incorporated by reference in their entirety.
Compounds such as IPI-145 and CAL130 have been reported as dual inhibitors of Pi3K δ/γ (WO2012/008302 & WO2012/121953 respectively), IPI-145 is under clinical investigation for cancer, asthma and rheumatoid arthritis. IPI-45 has been reported to have a maximum tolerated dose (MTD) of 75 mg BID (55th ASH® Annual Meeting. New Orleans-LA, Dec. 7-10, 2013). There are no reports of CAL-130 being investigated for clinical purposes.
There still remains an unmet need for dual δ/γ PI3K modulators for the treatment of diseases and disorders associated with δ/γ PI3K kinases-mediated events.
Further reference is made herein to International Publication Nos. WO 11/055215 and WO 12/151525 and U.S. Publication Nos. 2011/0118257 and 2012/0289496, each of which is incorporated herein by reference in its entirety.